RNA interference (RNAi) has become an important new tool for the targeted inhibition of gene expression. RNAi methodologies are accelerating basic medical research in numerous fields and have many potential clinical applications. However, there are still many gaps in our understanding of the basic mechanism of RNAi. The research proposed here is directed at understanding RNAi and improving related technologies for gene silencing. We now posses a basic outline of the core mechanism of RNAi, and an understanding of how related mechanisms are important in genome maintenance and developmental gene regulation in a variety of animals and plants. The proposed studies will address the following questions: (i) How are RNAi silencing mechanisms triggered? (ii) How do the RNAi and developmental pathways, which employ similar mechanisms, mediate distinct outcomes, mRNA destruction, translation inhibition and transcriptional silencing? and (iii) what additional gene products function in RNAi and related pathways? These questions will be addressed with an integrated set of molecular, biochemical and genetic methods using the nematode Caenorhabditis elegans as a model system. The findings from these studies will advance our understanding of RNAi and of related pathways in other organisms including humans and may lead to improved genetic interference technologies. Moreover, the genetic mechanisms of RNAi are related to ancient gene-regulatory mechanisms and are thus likely to be relevant to many aspects of human development and disease.